Subtypes H5, H7 and H9 of avian influenza virus, duck hepatitis virus, duck enteritis virus, duck Tembusu virus, Newcastle disease virus, egg drop syndrome virus, Muscovy duck reovirus, Muscovy duck parvovirus and duck circovirus are 11 kinds of major communicable diseases severely harm to duck. With the development of duck breeding, the incidence of duck communicable virus diseases is also gradually increasing, which has become an important factor which restricts the development of duck breeding. Traditional methods of differential diagnosing these duck communicable diseases mainly include isolation and identification of pathogen, serological tests etc., while these methods are often limited by freshness of clinical samples, pollution degree of clinical samples, or course of disease of clinical samples, the operations thereof are also very complicated, time-consuming, furthermore, it is more difficult for these methods to differential diagnose multiplex infections. In recent years, with the development of molecular biology, PCR technology has been widely used in detection of duck communicable diseases, and multiplex PCR detection technology has been established, which can detect several kinds of pathogens at the same time and needs several primer pairs to be mixed together and amplify competitively and simultaneously, while the interference will emerge between these primers, with addition of each primer pair, the sensitivity decreases. PCR products need to be observed by agarose gel electrophoresis which is difficult to distinguish bands within 50 bp-100 bp, and generally only duplex-sextuple PCRs can be carried out, so it is difficult to achieve high throughput detection. Multiplex fluorescent PCR generally can only detect quadruplex PCR, because probes need to be labeled with fluorescent groups with different light emitting wavelengths, if probes labeled with too many fluorescent groups, interference will emerge between each other, so multiplex fluorescent PCR can only detect duplex-quadruplex PCR. Because several primer pairs are present in the multiplex PCR reaction system at the same time, the possibility of forming complex primer dimers greatly increases, and target gene number which can be detected simultaneously is limited (usually 2-6 genes), all these reasons cause that the multiplex PCR cannot achieve the goal of high throughput and rapid detection and analysis.
GeXP system (Gene Expression Profiler Genetic Analysis System) is a platform developed by Beckman Coulter Company in the USA and used to study the quantitative analysis of multi-gene expression, GeXP system consisting of 2 parts: GeXP eXpression Profiler software used to design primers and capillary electrophoresis of GenomeLab™ GeXP Genetic Analysis System used to analyze results, of which the latter can well separate the adjacent amplification fragments with gaps of more than 7 bps. GeXP multiplex PCR amplification combines universal primers labeled with fluorescence markers and specific chimeric primers (namely sequences with 5′ ends of gene specific primers connecting to the universal primers) to cause the amplification of multiplex systems. At the beginning of the PCR reaction, reverse transcription reaction proceeds with the combination of reverse specific chimeric primer and original template, then the second chain of cDNA is synthetized by forward specific chimeric primer, after that, the specific sequences of forward and reverse chimeric primers initiate PCR reaction with cDNA as the template, complementary sequences of universal primers are respectively amplified; then universal primers labeled by fluorescence markers and dominated in the reaction system combine with complementary sequences thereof, causing subsequent amplification, and universal primers are complementary to base sequences labeled with fluorescence markers in the reaction system, PCR products are separated by GeXP capillary electrophoresis, and PCR products labeled with fluorescence markers are detected by GeXP detection window, then the lengths of the amplification fragments are calculated based on migration time of test fragments and standard molecular fragments (DNA Size Standard, DSS), the fluorescence signal intensity reflects the amplification content of the separated fragments. GeXP system can effectively analyze up to 40 target genes in the same system.
The key of establishing a detection method and making a detection kit for simultaneous identification of multiple pathogens with GeXP Multiplex Genes Expression Profiler Genetic Analysis System is to design specific primers and combine multiplex primers, then to use universal primers and convert the amplification of multiplex primers to the amplification of 1 pair of universal primer, so as to achieve the goal of high throughput detection. At present, there is no reagent or detection kit which can simultaneously detect multiple pathogens of communicable diseases from duck source based on GeXP system.